Abstract
Site-specific enzymatic reactions with microbial transglutaminase (mTGase) lead to a homogenous species of immunoconjugates with a defined ligand/antibody ratio. In the present study, we have investigated the influence of different numbers of 1,4,7,10-tetraazacyclododecane-N-N′-N′′-N′′′-tetraacetic acid (DOTA) chelats coupled to a decalysine backbone on the in vivo behavior of the chimeric monoclonal anti-L1CAM antibody chCE7agl. The enzymatic conjugation of (DOTA)1-decalysine, (DOTA)3-decalysine or (DOTA)5-decalysine to the antibody heavy chain (via Gln295/297) gave rise to immunoconjugates containing two, six or ten DOTA moieties respectively. Radiolabeling of the immunoconjugates with 177Lu yielded specific activities of approximately 70 MBq/mg, 400 MBq/mg and 700 MBq/mg with increasing numbers of DOTA chelates. Biodistribution experiments in SKOV3ip human ovarian cancer cell xenografts demonstrated a high and specific accumulation of radioactivity at the tumor site for all antibody derivatives with a maximal tumor accumulation of 43.6±4.3% ID/g at 24 h for chCE7agl-[(DOTA)-decalysine]2, 30.6±12.0% ID/g at 24 h for chCE7agl-[(DOTA)3-decalysine]2 and 49.9±3.1% ID/g at 48 h for chCE7agl-[(DOTA)5-decalysine)]2. The rapid elimination from the blood of chCE7agl-[(DOTA)-decalysine]2 (1.0±0.1% ID/g at 24 h) is associated with a high liver accumulation (23.2±4.6% ID/g at 24 h). This behavior changed depending on the numbers of DOTA moieties coupled to the decalysine peptide with a slower blood clearance (5.1±1.0 (DOTA)3 versus 11.7±1.4% ID/g (DOTA)5, p<0.005 at 24 h) and lower radioactivity levels in the liver (21.4±3.4 (DOTA)3 versus 5.8±0.7 (DOTA)5, p<0.005 at 24 h). We conclude that the site-specific and stoichiometric uniform conjugation of the highly DOTA-substituted decalysine ((DOTA)5-decalysine) to an anti-tumor antibody leads to the formation of immunoconjugates with high specific activity and excellent in vivo behavior and is a valuable option for radioimmunotherapy and potentially antibody-drug conjugates (ADCs).
Highlights
One of the remaining challenges of immunoconjugation is product homogeneity with regard to site-specificity and stoichiometry of antibody modification [1,2,3]
We have demonstrated that it is possible to use microbial transglutaminase to site- modify the chimeric IgG1-type tumor-targeting anti-L1CAM antibody chCE7agl with various numbers of DOTA metal chelating systems attached to a polylysine backbone
This method allowed the production of homogenous immunoconjugate with a precise and predictable number of metal chelating agents per antibody
Summary
One of the remaining challenges of immunoconjugation is product homogeneity with regard to site-specificity and stoichiometry of antibody modification [1,2,3]. A possible explanation is that the body recognizes highly substituted immunoconjugates as a damaged form of the protein and quickly clears them from the blood [7]. It is common for randomly conjugated antibodies to be modified in positions that weaken or even abrogate antigen binding, which in turn decreases the efficacy of the targeting immunoconjugate [8,9,10]. It has been observed that chemically substituted radioimmunoconjugates with high numbers of metal chelators show higher uptake and retention in non-targeted organs and tissues while often clearing faster from the blood pool, resulting in poor target/non-target ratios [12,13,14]
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